TY - JOUR
T1 - Isolation of human DNA-unwinding elements as sites of DNA polymerase α/primase entry
AU - Pack, Robert A.
AU - Toshiki, Tsurimoto
N1 - Funding Information:
for the collaboration to construct the plasmid library of the MENZA region. for providing us with ten plasmids containing randomly cloned human DNA and for their helpful discussions. We are grateful to Dr. K. Shirahige (Osaka University, Osaka, Japan) for the calculation of the AC values for the stability of the DNA duplex, and to T. Oku for his technical assistance. This work was supported by grants from the Ministry of Education, Science and Culture, Japan and the Asahi Glass Foundation.
PY - 1994/10/21
Y1 - 1994/10/21
N2 - Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.
AB - Human DNA libraries were screened for DNA synthesis activity in vitro using purified DNA polymerase α/primase and a viral DNA helicase (simian virus 40 large tumor antigen). Three clones exhibited a high activity distinguishable from the rest. The DNA synthesis was dependent on negative supertwisting and initiated at a unique region in the human DNA insert. Functional subclone DNA fragments which could be shortened to less than 1 kb are located in the initiation region. Binding with a single-stranded DNA-binding protein and digestion with nuclease P1 demonstrated that these DNAs have a highly single-stranded nature at a certain site in a closed circular plasmid. The minimal functional sequences coincide with the single-stranded region and contain a characteristic dinucleotide repeat sequence. These repeats have an extremely low free energy for DNA strand separation and are denned as DNA-unwinding elements, which are frequently observed at regions flanking replication origins in Escherichia coli and Saccharomyces cerevisiae chromosomes. We suggest that such a repeating sequence would have an important role during initiation of DNA replication and function as a site to recruit replication proteins. DNA replication; DNA helicase; dinucleotide repeats; replication protein A; nuclease P1.
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U2 - 10.1016/0378-1119(94)90699-8
DO - 10.1016/0378-1119(94)90699-8
M3 - Article
C2 - 7958955
AN - SCOPUS:0027959908
SN - 0378-1119
VL - 148
SP - 277
EP - 284
JO - Gene
JF - Gene
IS - 2
ER -